cathelicidin deficiency predisposes to eczema herpeticum
TRANSCRIPT
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Food allergy, dermatologic diseases, and anaphylaxis
Cathelicidin deficiency predisposes toeczema herpeticum
Michael D. Howell, PhD,a Andreas Wollenberg, MD,b Richard L. Gallo, MD, PhD,c
Michael Flaig, MD,b Joanne E. Streib, BA,a Cathy Wong, BS,c Tatjana Pavicic, MD,b
Mark Boguniewicz, MD,a and Donald Y. M. Leung, MD, PhDa Denver, Colo, Munich,
Germany, and San Diego, Calif
Background: The cathelicidin family of antimicrobial peptides
is an integral component of the innate immune response that
exhibits activity against bacterial, fungal, and viral pathogens.
Eczema herpeticum (ADEH) develops in a subset of patients
with atopic dermatitis (AD) because of disseminated infection
with herpes simplex virus (HSV).
Objective: This study investigated the potential role of
cathelicidins in host susceptibility to HSV infection.
Methods: Glycoprotein D was measured by means of
real-time RT-PCR as a marker of HSV replication in skin
biopsy specimens and human keratinocyte cultures.
Cathelicidin expression was evaluated in skin biopsy
specimens from patients with AD (n 5 10) without a
history of HSV skin infection and from patients with ADEH
(n 5 10).
Results: The cathelicidin peptide LL-37 (human cathelicidin)
exhibited activity against HSV in an antiviral assay, with
significant killing (P < .001) within the physiologic range. The
importance of cathelicidins in antiviral skin host defense was
confirmed by the observation of higher levels of HSV-2
replication in cathelicidin-deficient (Cnlp2/2) mouse skin
From athe Division of Allergy and Immunology, Department of Pediatrics,
National Jewish Medical and Research Center, and the Department of
Pediatrics, University of Colorado Health Sciences Center, Denver; bthe
Department of Dermatology and Allergy, Ludwig Maximilian’s University,
Munich; cthe Division of Dermatology, Department of Medicine and Pedi-
atrics, University of California at San Diego and VA San Diego Health Care
System.
Supported in part by the National Institutes of Health National Research Ser-
vice Award (T32 AI 07365) and AAAAI Fujisawa Skin Diseases Award
(M.D.H.); NIH grants AI052453 and AR45676 and a VA Merit Award
(R.L.G.); and NIH grants AR41256 and 5R21AR051634, NIH/NIAID con-
tracts N01 AI40029 and N01 AI40030, General Clinical Research Center
grant MO1 RR00051 from the Division of Research Resources, the Edel-
stein Family Chair in Pediatric Allergy and Immunology, and the University
of Colorado Cancer Center (D.Y.M.L.).
Disclosure of potential conflict of interest: R. Gallo has consultant arrange-
ments with Ceragenix. M. Boguniewicz has received grants from Novartis
and Astellas and is lecture honoraria for Novartis and Astellas. All other
authors—none disclosed.
Received for publication October 28, 2005; revised December 6, 2005;
accepted for publication December 7, 2005.
Available online February 14, 2006.
Reprint requests: Donald Y. M. Leung, MD, PhD, National Jewish Medical
and Research Center, 1400 Jackson St, Room K926i, Denver, CO 80206
E-mail: [email protected].
0091-6749/$32.00
� 2006 American Academy of Allergy, Asthma and Immunology
doi:10.1016/j.jaci.2005.12.1345
836
(2.6 6 0.5 pg HSV/pg GAPDH, P < .05) compared with that
seen in skin from their wild-type counterparts (0.9 6 0.3).
Skin from patients with ADEH exhibited significantly
(P < .05) lower levels of cathelicidin protein expression than
skin from patients with AD. We also found a significant
inverse correlation between cathelicidin expression and
serum IgE levels (r2 5 0.46, P < .05) in patients with AD
and patients with ADEH.
Conclusion: This study demonstrates that the cathelicidin
peptide LL-37 possesses antiviral activity against HSV and
demonstrates the importance of variable skin expression
of cathelicidins in controlling susceptibility to ADEH.
Additionally, serum IgE levels might be a surrogate
marker for innate immune function and serve as a
biomarker for which patients with AD are susceptible to
ADEH.
Clinical implications: A deficiency of LL-37 might render
patients with AD susceptible to ADEH. Therefore
increasing production of skin LL-37 might prevent herpes
infection in patients with AD. (J Allergy Clin Immunol
2006;117:836-41.)
Key words: Antimicrobial peptides, herpes simplex virus, atopicdermatitis, eczema herpeticum
Atopic dermatitis (AD) is a chronic inflammatoryskin disease that affects approximately 17% of childrenand is associated with recurrent skin infections.1,2
Recent studies have shown that the innate immune res-ponse and, more specifically, antimicrobial peptides(AMPs) are decreased in the skin of patients with AD.It has been postulated that this might explain the sus-ceptibility of these individuals to recurrent bacterialskin infections.3,4
Eczema herpeticum (ADEH) is a disseminated herpessimplex virus (HSV) 1 or 2 infection that occurs in a subsetof patients with AD.5,6 Left untreated, ADEH might befatal because of systemic viremia.7 However, only a smallsubset of patients with AD have problems with recurrentviral infections, suggesting they might have an AMPphenotype distinct from that of most patients with AD.Therefore this study was conducted to investigate therole of human cathelicidin (LL-37) in controlling HSVinfection and to examine differences in cathelicidinexpression between patients with uncomplicated AD andpatients with ADEH.
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Abbreviations used
AD: Atopic dermatitis
ADEH: Eczema herpeticum
AMP: Antimicrobial peptide
CRAMP: Mouse cathelicidin
HSV: Herpes simplex virus
LL-37: Human cathelicidin
MEM: Minimal essential medium
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,
5-Diphenyltetrazolium bromide
pfu: Plaque-forming units
METHODS
Patients
Study participants included 10 patients with AD without a history
of ADEH (mean age, 29.3 6 6.9 years) and 10 patients with AD with
a history of ADEH (mean age, 39.8 6 4.2 years). Patients were
classified as having ADEH on the basis of clinical signs of ADEH, as
diagnosed by a dermatologist, and a confirmation of HSV infection
by means of either PCR or serology. Total serum IgE levels were
measured with the UniCAP system (Pharmacia, Uppsala, Sweden).
Patients in these studies were never taking oral steroids or systemic
calcineurin inhibitors and stopped taking topical calcineurin inhib-
itors for a minimum of 1 week before enrollment. These studies were
conducted according to the Declaration of Helsinki Guidelines
and approved by the institutional review board at National Jewish
Medical and Research Center in Denver and Ludwig Maximilian’s
University in Munich. All patients provided written informed consent
before participation in these studies.
Skin biopsy specimens were collected from the lesional eczema-
toid skin rash of patients with AD and ADEH. After collection, skin
biopsy specimens were fixed in formalin and archived.
Mice
BALB/c mice were purchased from the Jackson Laboratory (Bar
Harbor, Me). Cnlp2/2 (Cnlp knockout) mice were obtained from
R. L. Gallo (Veterans Affairs Medical Center and the University of
San Diego, San Diego, Calif) and backcrossed onto the BALB/c
background. All protocols with these animals were approved by the
Institutional Animal Care and Use Committee at National Jewish
Medical and Research Center. This institution has an animal welfare
assurance number (A3026-1) on file with the Office of Protection and
Research Risks.
Preparation of virus
Human herpes virus type 2 (HSV-2; a gift from Dr Adriana
Weinberg, University of Colorado Health Science Center) was grown
and passaged in human embryonic lung fibroblasts in Earle’s minimal
essential medium (MEM; GIBCO, Grand Island, NY) with 2.5% FCS
(Gemini Bio Products, Woodland, Calif) and antibiotics. Freshly
trypsinized lung fibroblasts were grown for 3 days to confluence and
inoculated with approximately 1 plaque-forming unit (pfu) per cell in
culture medium. Cells were checked daily for cytopathic effects. The
culture supernatant was harvested after 48 to 72 hours of incubation at
37�C in 5% CO2, freeze-thawed 5 times, and centrifuged for 15 min-
utes at 1000 rpm. For virus titration, 10-fold dilutions of stock were
made, and 0.1 mL of each dilution was added to the fibroblast cell
sheets in 24-well tissue-culture plates. Adsorption was allowed to
take place for 1 hour at 37�C in 5% CO2 and was followed by the
addition of Earle’s MEM with 2.5% FCS. Forty-eight hours after
infection, medium was removed, and cells were fixed with formalin-
crystal violet. Plaques were visualized on an Inverted Nikon Micro-
scope (Nikon, Tokyo, Japan) under 1.3 3 10 magnification. Virus
stocks were stored at 270�C.
Peptide preparations
Human cathelicidin (LL-37) and an irrelevant control peptide,
8044 (GLNGPDIYKGUYQFKSVEFD), were synthesized by means
of solid-phase t-BOC chemistry with standard methodology and
purified to homogeneity through highly purified liquid chromatog-
raphy by the Molecular Resource Center at National Jewish. Peptide
8044 was chosen from a library of existing peptides for use as a
control having no sequence identity with the test peptide. The iden-
tity of LL-37 was confirmed by means of mass spectroscopy.
Concentrations of LL-37 and control peptides used in these experi-
ments ranged from 0 to 100 mM.
Viral killing assay
BS-C-1 African green monkey kidney cells were seeded at 2 3
105 cells/well in 24-well plates (Becton Dickinson, Torreyana, Calif)
and allowed to grow to confluence overnight at 37�C in 5% CO2 in
Earle’s MEM with 10% FCS and antibiotics. To examine the effects
of LL-37 and control peptide, 0 to 100 mM was incubated with 2 3
103 pfu HSV-2 for 24 hours at 37�C in a volume not to exceed 0.1 mL.
Growth medium was removed from the cell sheet and rinsed once
with Earle’s MEM with 2.5% FCS. The virus-protein complex was
added to the cells and adsorbed for 1 hour at 37�C in 5% CO2.
Growth medium was added to 0.5 mL and incubated for 24 hours
for RNA analysis of HSV gene expression and 48 hours for plaque
development. For the plaque assay, the medium was removed, and
wells were overlaid with 0.5 mL of 4% buffered formalin and allowed
to fix for 10 minutes at room temperature. The formalin was removed,
and 0.5 mL of 0.1% crystal violet in PBS was added to the wells for
5 minutes at room temperature. Wells were then aspirated and air-
dried for visualization of plaques. We found the most accurate results
with the virus alone forming 50 to 80 plaques per well.
Keratinocyte cell culture
HaCaT cells, a human keratinocyte cell line, were cultured in
Dulbecco’s modified Eagle’s medium (Cellgro, Herdon, Va) supple-
mented with 10% FCS (Gemini Bio Products) and 1% of the
following until confluent: penicillin-streptomycin, l-glutamine, MEM
with nonessential amino acids (GIBCO), and MEM vitamins solution
(GIBCO).
HaCaT cells were infected with 0.05 pfu/cell of HSV-2 for 6 hours
to evaluate the antiviral activity of LL-37. After the incubation,
HSV-2 was removed, and cells were washed with medium to remove
remaining HSV-2. LL-37 (0-100 mM) was added to the cells and
allowed to incubate for an additional 18 hours. RNA was isolated
from the cells for analysis of HSV-2 gene expression.
Murine skin explant cultures
The dorsal thorax of all mice was clipped and treated with the
depilatory agent Nair to remove hair. Seventy-two hours after hair
removal, mice were killed by means of CO2 asphyxiation. Six-milli-
meter punch biopsy specimens were collected from the dorsal thorax
and immediately placed in a 96-well plate and RPMI (Cellgro) sup-
plemented with 10% FCS (Gemini Bio Products). Murine skin biopsy
specimens were cultured in the presence of media alone or 2 3 104
pfu HSV-2 for 24 hours. After the exposure period, medium was
removed, and biopsy specimens were submerged in Tri-Reagent
(Molecular Research Center Inc, Cincinnati, Ohio) for RNA isola-
tion. Three independent experiments were conducted, with a total
of 15 mice in each exposure group. Data from one representative
experiment are shown.
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Real-time RT-PCR
Total RNA was isolated from skin biopsy specimens by means
of chloroform-phenol extraction and isopropanol precipitation, ac-
cording to the manufacturer’s guidelines (Molecular Research Center
Inc). RNeasy Mini Kits (Qiagen, Valencia, Calif) were used,
according to the manufacturer’s protocol, to isolate RNA from cell
cultures and to further purify RNA from skin biopsy specimens.
Real-time RT-PCR was performed with an ABI 7000 Sequence
Detection system (Applied Biosystems, Foster City, Calif), as
previously described.4 Human GAPDH and rodent GAPDH were
purchased from Applied Biosystems. Primer and probe sequences
for cathelicidin were designed as previously described.3 Primer and
probe sequences used to assay HSV-2 gene transcripts were as fol-
lows: forward, 59- CGC TCT CGT AAA TGC TTC CCT -39; reverse,
59- TCT ACC CAC AAC AGA CCC ACG -39. This region of the
genome encodes glycoprotein D of HSV-2.8 Relative expression
levels were calculated by using the relative standard curve method,
as outlined in the manufacturer’s technical bulletin. Quantities of
all targets in test samples were normalized to the corresponding
GAPDH or total RNA levels and expressed as target gene normalized
to GAPDH or target gene normalized to total RNA to allow for
comparisons between samples and groups. A standard curve was
generated with cDNA from purified herpes virus.
FIG 1. LL-37 exhibits antiviral activity against HSV. Physiologic
concentrations of LL-37 were preincubated with HSV-2 for 24 hours
and then added to BS-C-1 for an additional 24 hours to evaluate
HSV-2 gene expression by means of real-time RT-PCR (A) or 48
hours to investigate functionally active virus by using a standard
plaque assay (B). ***Significant difference of P < .001 compared
with 0 mM.
Cathelicidin protein expression
Paraffin-embedded tissues were cut into 5-mm sections, deparaffi-
nized, rehydrated, and then stained with rabbit anti-LL-37 (5 mg/mL),
as previously described.3 All slides were coded to ensure patient an-
onymity, and readings were done blind so that the slide reader was
unaware of the identity of the slides. Images were collected at 403
magnification, and the intensity of the immunostaining was scored
on a scale from 0 to 5, with 0 indicating no staining and 5 indicating
the most intense staining.
Statistical analyses
All statistical analysis was conducted with GraphPad Prism,
version 3.01 (San Diego, Calif). Statistical differences in gene
expression or protein staining between multiple groups was
determined by using a 1-way ANOVA, and significant differences
were determined by using the Tukey-Kramer test.9 Statistical dif-
ferences in total serum IgE levels were determined by using a
Student t test.
RESULTS
Anti-HSV activity of LL-37
In our initial experiments we examined whether LL-37(0-100 mM) could directly kill HSV. As shown in Fig 1, A,we observed a concentration-dependent inhibition of viralreplication measured by means of real-time RT-PCR.Significant reduction in viral replication by LL-37 was ob-served with concentrations as low as 5 mM (mean, 2444 6
1223 ng HSV/ng total RNA; P < .001) compared withHSV alone (mean, 32,620 6 4061 ng HSV/ng totalRNA). This was confirmed by using a standard viral pla-que assay in which preincubation of HSV with 5 mM ofLL-37 significantly (P < .001) reduced the number ofplaques from 78.3 6 1.5 (HSV alone) to 40.3 6 1.9(Fig 1, B). The control peptide 8044 possessed no antiviralactivity against HSV.
Role of cathelicidins in controlling HSVreplication in the skin
To examine a more physiologic condition, humankeratinocyte cultures were preinfected with HSV for 6hours and then treated with exogenous LL-37 to determinewhether intracellular viral replication could be halted withphysiologic concentrations of LL-37. Fig 2 demonstratesthat concentrations of LL-37 as low as 25 mM (mean,563 6 71 ng HSV/ng GAPDH) were able to significantly(P < .01) reduce the levels of HSV gene expression in pre-viously infected keratinocytes (HSV mean, 1614 6 158).
To demonstrate the clinical relevance of LL-37 com-pared with other potential arms of the innate immuneresponse in limiting HSV infection, we used mice defi-cient in Cnlp, the murine cathelicidin. Significantly higherlevels of HSV replication were observed in skin biopsyspecimens from Cnlp knockout mice (BALB/c back-ground; 2.6 6 0.5 pg HSV/pg GAPDH, P < .05) com-pared with that seen in skin biopsy specimensfrom wild-type BALB/c mice (0.9 6 0.3 pg HSV/pgGAPDH, Fig 3), suggesting that cathelicidins play an
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important role in controlling HSV skin infection. Thesedata are representative of 3 independent experimentswith a total of 15 mice.
Deficiency of LL-37 in the skin of patientswith ADEH
Skin biopsy specimens were collected from the skinlesions of adult patients with AD and patients with ADEH.Biopsy specimens were stained with a polyclonal antibodyspecific for LL-37 to investigate cathelicidin expression.All slides were coded before analysis, and readings weredone blind so that the slide reader was unaware of theidentity of the slides. Fig 4, A, shows that skin sectionsfrom patients with AD exhibited more staining for cathe-licidin than skin lesions from patients with ADEH. Thecomposite data for cathelicidin immunostaining in allsamples is shown in Fig 4, B. The intensity of cathelicidinstaining in ADEH skin lesions was significantly (P < 0.05)lower than that seen in skin lesions from patients with AD.
Correlation between serum IgE leveland cathelicidin expression
Previous studies have suggested that TH2 cytokinescould inhibit cathelicidin production.10 Because of thelimited amount of archived tissue available, we wereunable to investigate potential differences in IL-4 andIL-13 expression between patients with AD and patientswith ADEH. However, IL-4 and IL-13 are TH2 cytokinesessential in the production of IgE.11 Therefore we exam-ined whether increases in serum IgE levels might correlatewith cathelicidin expression in patients with ADEH andpatients with AD. Because antibody generation is expo-nential, we log transformed the serum IgE values forfurther statistical analysis. Using linear regression analy-sis, we demonstrate a significant correlation (r2 5 0.46,P < .05) between total serum IgE levels and cathelicidin
FIG 2. Exogenous LL-37 rescues HSV-infected keratinocytes.
Human keratinocytes were infected with 0.05 pfu/cell HSV-2 for
6 hours and then treated with physiologic concentrations of LL-37
for an additional 18 hours. RNA was isolated from the cells, and
the levels of HSV-2 gene expression were evaluated by means of
real-time RT-PCR. **Significant difference of P < .01 compared
with HSV alone.
protein expression in patients with AD and patients withADEH (Fig 5).
DISCUSSION
AMPs are an integral part of the innate immuneresponse because they have been shown to be effectivein killing bacterial3 and viral12 pathogens. Cathelicidin isproduced by several cells in the skin, including keratino-cytes, where it is induced in response to inflammatorystimuli.13,14 On release, the cathelicidin precursor proteinis processed into the biologically active AMP LL-37. Inthis study we demonstrate that LL-37 exhibits antiviralactivity against HSV. Our previous studies have demon-strated that patients with AD, in general, have a reducedability to generate cathelicidin in their skin comparedwith patients with psoriasis or allergic contact dermatitis,and this might predispose them to microbial skin infec-tion.3,10 However, the propensity of patients with AD tohave serious skin infection, such as ADEH, has not previ-ously been explored. Results from the current study indi-cate that a more exaggerated reduction in cathelicidinexpression might predispose a subset of patients withAD to having ADEH. Thus there is heterogeneity in theexpression of cathelicidin within AD, such that the indi-viduals with the lowest levels are most prone to dissemi-nated viral infection.
It was previously reported that LL-37 exhibits littleactivity against HSV-1 or HSV-2.15 In contrast, we dem-onstrate in this study that LL-37 exhibits potent antiviralactivity against HSV. In the previous study Yasin et al15
demonstrated that 44.5 mM of LL-37 provided 28% and46% protection against HSV-1 and HSV-2, respectively,using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) cytotoxicity assay. In the MTTassay antiviral activity of LL-37 is determined on the basis
FIG 3. Essential role of cathelicidins in controlling HSV replication
in the skin. Skin biopsy specimens from BALB/c (n 5 5) and Cnlp
knockout (n 5 5) mice were stimulated with HSV-2 for 24 hours and
evaluated for HSV-2 gene expression. RNA was collected from the
tissue, and the levels of HSV-2 were evaluated by means of real-
time RT-PCR. *Significant difference of P < .05.
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FIG 4. Expression of cathelicidin increased in AD skin compared with that seen in ADEH skin. A, Paraffin-embed-
ded skin explants from patients with AD (n 5 10) and patients with ADEH (n 5 10) were cut into 5-mm sections
and stained for human cathelicidin. B, The intensity of the immunostaining was visually scored on a scale from 0
to 5, with 0 indicating no staining and 5 indicating the most intense staining. *Significant difference of P < .05.
of increases in cellular proliferation compared with cellstreated with virus alone. In our current study we measuredHSV replication by evaluating glycoprotein D geneexpression using real-time RT-PCR. Directly measuringthe virus activity rather than cellular proliferation providesa more direct and appropriate measurement of the antiviralactivity of LL-37 against HSV, accounting for differencesbetween our results and those of Yasin et al.15 Our obser-vation is further supported by Gordon et al,16 who recentlydemonstrated that LL-37 exhibits antiviral activity againstHSV-1 in corneal and conjunctival epithelia.
In this study we demonstrate that concentrations aslow as 10 mM of LL-37 reduce HSV levels by more than10,000-fold. Because psoriatic skin can contain up to 1605mM LL-37,3 this demonstrates that physiologic concen-trations of LL-37 are effective at controlling HSV rep-lication. This was further supported by using a morephysiologic approach in which keratinocytes were prein-fected with HSV for 6 hours and then incubated withLL-37 for an additional 18 hours. Again, we saw greaterthan a 60% reduction in the levels of HSV when as littleas 25 mM of LL-37 was added to previously infectedkeratinocytes. The importance of cathelicidins in skin
innate immune responses to HSV is also strongly sup-ported by our current finding that skin explants frommice deficient in the cathelicidin gene Cnlp and its AMPproduct, mouse cathelicidin (CRAMP), sustain higherlevels of HSV replication after inoculation comparedwith those seen in their wild-type counterparts. MouseCRAMP is very similar to human LL-37 in structure, tis-sue distribution, and antimicrobial activity and is thereforea reasonable model of the human cathelicidin. The obser-vation that CRAMP-deficient mice support a higher levelof HSV replication reinforces the important effect that acathelicidin deficiency would have on HSV skin infectionin human subjects. These data support a conclusion thatdecreased cathelicidin expression will significantly in-crease the potential for disseminated skin infection tooccur.
Because not all patients with AD have ADEH, weinvestigated the abundance of cathelicidin in the skin ofpatients with AD and patients with ADEH to determinewhether the development of ADEH corresponded withdecreased cathelicidin expression. Skin biopsy specimenswere obtained from naturally induced inflammatory skinrashes of AD and ADEH. This allowed for comparisons
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between similarly stimulated skin samples. Lesional skinfrom patients with ADEH exhibited significantly lowerlevels of cathelicidin protein than skin lesions of patientswith uncomplicated AD. Our results suggest that patientswith AD with the lowest levels of cathelicidin are mostsusceptible to development of ADEH and that lack of thismolecule might serve as a biomarker for patients at risk fordisseminated viral skin infection.
AD skin is characterized by the overexpression of theTH2 cytokines IL-4 and IL-13.17 Previously, we demon-strated that TH2 cytokines downregulate cathelicidin.10
However, because of the difficulty in identifying patientswith ADEH and insufficient amounts of archived tissue,we were unable to investigate the levels of IL-4 and IL-13 in patients with AD and patients with ADEH for poten-tial differences. IL-4 and IL-13 are TH2 cytokines essentialin the production of IgE.11 Therefore the measurementof serum IgE levels might serve as a biomarker for levelsof TH2 responses. Wollenberg et al6 and Lagace-Simardet al18 have previously demonstrated that patients withADEH exhibited higher total serum IgE levels thanpatients with AD. We confirmed this observation in thecurrent study by demonstrating significantly higher serumIgE levels in patients with AD compared with those seenin patients with ADEH. We further determined that thereis a strong correlation between the levels of serum IgEand cathelicidin protein expression in patients with ADand patients with ADEH. Therefore serum IgE levelsmight serve as a surrogate marker for the expression ofcathelicidin in the skin of patients with AD and patientswith ADEH and might separate those who are more sus-ceptible to disseminated viral infection.
The current study therefore demonstrates that ADrepresents a heterogeneous population of patients express-ing different levels of cathelicidin in the skin. Thisexplains, in part, why a subgroup of patients with AD issusceptible to ADEH after HSV infection. Additionally,this study demonstrates the importance of the cathelicidin
FIG 5. Correlation between serum IgE levels and cathelicidin
expression in patients with AD and patients with ADEH. Serum
IgE levels were determined from patients with AD (n 5 9) and
patients with ADEH (n 5 9). Regression analysis was performed on
log-transformed serum IgE values and LL-37 protein expression.
in controlling the replication of HSV in the skin. This issupported by significantly higher levels of HSV replica-tion in the skin of Cnlp knockout mice and the significantreduction of HSV gene expression in keratinocytes treatedwith LL-37. Overall, these data suggest further clinicalstudies are warranted to examine whether augmentationof LL-37 expression in AD skin might be useful inthe prevention of ADEH and the perplexing challenge ofcontrolling microbial infection in this common skinproblem.
We thank Dr Adriana Weinberg at the University of Colorado
Health Sciences Center for the contribution of the human embryonic
lung fibroblasts and HSV. We also thank Maureen Sandoval for her
assistance in the preparation of this manuscript and the nursing staff
in the General Clinical Research Center for their help in patient
recruitment and sample collection.
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